The Lack of NK Cytotoxicity Associated with Fresh HUCB May Be Due to the Presence of Soluble HLA in the Serum

Abstract

Human bone marrow transplantation is becoming more common in the treatment of certain forms of cancer despite the scarcity of HLA matched donors. Because human umbilical cord blood (HUCB) has been used as a source for stem cells in bone marrow transplantation, and because NK cells appear to be important in graft versus leukemia response, we investigated the lytic activity of freshly isolated HUCB NK cells (HUCB-NK) against tumor targets and their ability to differentiate into LAK cells following stimulation with various cytokines. Although cytotoxicity mediated by fresh HUCB-NK was low compared to that of adult peripheral blood lymphocyte-derived NK cells (PBL-NK), the ability of HUCB-NK to bind to K562 target cells (TC) was similar to PBL-NK. In addition, the PBL-NK cytotoxicity of postpartum mothers was also low compared to that of normal adult PBL-NK. When we incubated HUCB for 18 hr in either IL-2 or IL-12, we boosted the level of HUCB-NK cytotoxicity to approximately the level observed in PBL-NK and increased the level of perforin, granzyme A, and granzyme B mRNA expression. In addition, when we incubated HUCB in IL-2, IL-4, IL-7, IL-12, TNF-α, IFN-α, IFN-γ, or TGF-β for 5 days, we observed that HUCB was capable of generating LAK cells only when incubated with either IL-2 or IL-12. In contrast, IL-2, IL-7, IL-12, TNF-α, and IFN-γ all generated LAK cells from adult PBL. When we added to the medium low-dose IL-2 and irradiated K562 as feeder cells (mini-LAK), we were unable to generate LAK activity from HUCB-NK, whereas we could generate it with PBL-NK cells under the same conditions. Addition of serum derived from HUCB in a 4-hr51Cr release assay with PBL-NK as the effector cells (EC) and K562 as the TC resulted in a 42% decrease in PBL-NK-mediated cytotoxicity. Although we detected no TGF-β in HUCB serum, we did detect high concentrations of soluble class 1 MHC (sHLA). To our knowledge, sHLA has not previously been shown to inhibit NK cytotoxicity, although the expression of class 1 HLA on the surface of TC has been shown to inhibit NK cytotoxicity. To study further the effect of sHLA on cell-mediated cytotoxicity, we added various concentrations of sHLA to EC mediating NK, ADCC, and CTL activities. All were inhibited in a dose-dependent manner. Taken together, these results demonstrate that (i) although fresh HUCB-NK are functionally immature in that they fail to lyse TC, they acquire lytic potential only with IL-2 and IL-12; (ii) the increase in lytic potential is accompanied by an increase in the level of three lytic moieties known to mediate cytotoxicity, perforin, granzyme A, and granzyme B; (iii) the presence of sHLA class 1 in the serum of HUCB may be partially responsible for the lack of NK lytic activity.